Phosphorous (P) is an essential element for growth. A substantial amount of the phosphorous found in conventional livestock feed, e.g., cereal grains, oil seed meal, and by products that originate from seeds, is in the form of phosphate which is covalently bound in a molecule know as phytate (myo-inositol hexakisphosphate). The bioavailability of phosphorus in this form is generally quite low for non-ruminants, such as poultry and swine, because they lack digestive enzymes for separating phosphorus from the phytate molecule.
Several important consequences of the inability of non-ruminants to utilize phytate may be noted. For example, expense is incurred when inorganic phosphorus (e.g., dicalcium phosphate, defluorinated phosphate) or animal products (e.g., meat and bone meal, fish meal) are added to meet the animals' nutritional requirements for phosphorus. Additionally, phytate can bind or chelate a number of minerals (e.g., calcium, zinc, iron, magnesium, copper) in the gastrointestinal tract, thereby rendering them unavailable for absorption. Furthermore, most of the phytate present in feed passes through the gastrointestinal tract, elevating the amount of phosphorous in the manure (see, e.g., Common et al., 1989). This leads to an increased ecological phosphorous burden on the environment (See, e.g., Cromwell et al., 1991).
Ruminants, such as cattle, in contrast, readily utilize phytate thanks to an enzyme produced by rumen microorganisms known as phytase. Phytase catalyzes the hydrolysis of phytate to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate. Two different types of phytases are known: (1) a so-called 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and (2) a so-called 6-phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26). The 3-phytase preferentially hydrolyzes first the ester bond at the 3-position, whereas the 6-phytase preferentially hydrolyzes first the ester bond at the 6-position.
Microbial phytase, as a feed additive, has been found to improve the bioavailability of phytate phosphorous in typical non-ruminant diets (See, e.g., Cromwell, et al, 1993). The result is a decreased need to add inorganic phosphorous to animal feeds, as well as lower phosphorous levels in the excreted manure (See, e.g., Komegay, et al, 1996).
Despite such advantages, few of the known phytases have gained widespread acceptance in the feed industry. The reasons for this vary from enzyme to enzyme. Typical concerns relate to high manufacture costs and/or poor stability/activity of the enzyme in the environment of the desired application. A number of enzymatic criteria must be fulfilled by a phytase if it is to be attractive for widespread use in the animal feed industry. These include a high overall specific activity, a low pH optimum, resistance to gastrointestinal proteases and thermos/ability (See, e.g., Simons et al., 1990). Thermostability is perhaps the single most important prerequisite for successful application of feed enzymes because all of the components, including phytase, are briefly exposed to temperatures between 60 and 95° C. in the feed pelleting process. Since all known microbial phytases unfold at temperatures between 56 and 78° C. (Lehmann et al., 2000), genetically engineered enzymes that can overcome this limitation would clearly have an advantage in feed applications
It is, thus, generally desirable to discover and develop novel enzymes having good stability and phytase activity for use in connection with animal feed, and to apply advancements in fermentation technology to the production of such enzymes in order to make them commercially viable. It is also desirable to ascertain nucleotide sequences which can be used to produce more efficient genetically engineered organisms capable of expressing such phytases in quantities suitable for industrial production. It is still further desirable to develop a phytase expression system via genetic engineering which will enable the purification and utilization of working quantities of relatively pure enzyme.
The E. coli appA gene encodes a periplasmic enzyme that exhibits both acid phosphatase and phytase activity (see, Greiner et al.,1993). Based on a survey of purified phytases from several microbial sources, the native enzyme from E. coli exhibits the highest reported activity (see, Wyss et al.,1999). Furthermore, the enzyme exhibits a single pH optimum of 4.5 for phytase activity and a temperature optimum of ˜60° C. Therefore, based on its established high intrinsic activity, low pH optimum, and inherent temperature resistance, the E. coli phytase represents an excellent starting point from which to begin directed evolution of a thermostable phytase for commercial feed and various other phytase applications.
The DNA sequence of the complete E. coli K-12 appA gene was originally reported by Dassa et al. (1990). Since that original report, however, a number of appA gene variants (naturally occurring or laboratory generated) have been described. Ostanin et al. (1992) used site-directed mutagenesis to examine the catalytic importance of 2 histidine and 4 arginine residues which are conserved in a number of acid phosphatases. The replacement of Arg16(R16A) or His17 (H17N) within the conserved N-terminal RHGXRXP (SEQ ID No. 30) motif of the E. coli AppA protein completely abolished activity on p-nitrophenyl phosphate (pNPP). Mutagenesis of Arg20 (R20A), Arg92 (R92A) and His303 (H303A) resulted in proteins with only 0.4% the activity of the wild-type (WT) enzyme while replacement of Arg63 (R63A) did not affect activity. Site directed mutagenesis experiments, designed to explore the role of Asp304 as a proton donor, demonstrated only small decreases in Km values of the substrate pNPP for the mutants D304A and D304Q (Ostanin et al., 1993). However, Vmax values were greatly reduced.
Several bacterial strains isolated from the contents of pig colon were found to produce phytase activity. One strain, identified as an E. coli, had the highest activity. The appA gene (designated appA2) from this E. coli strain was found to be 95% identical with the E. coli K-12 appA gene sequence (Rodriguez et al., 1999). This corresponded to a six amino acid differences between the two proteins: S102P, P195S, S197L, K202N, K298M and T299A. The purified AppA2 protein (expressed in Pichia pastoris), however, had a dramatically lower activity than the purified AppA enzyme (Greiner et al., 1993; Wyss et al., 1999; Golovan et al., 2000).
Wild-type AppA and several mutants generated by site directed mutagenesis were expressed in P. pastoris to investigate the effect of N-linked glycosylation on the thermostability profile of the AppA protein (Rodriguez et al., 2000). AppA mutants A131N/V143N/D207N/S211N, C200N/D207N/S211N and A131N/V134N/C200N/D207N/S211N were examined for levels of glycosylation and phytase activity. Despite no enhancement of glycosylation, mutant C200N/D207N/S211N was more active at pH 3.5-5.5 and retained more activity after heat treatment than the WT protein produced in P. pastoris. In addition, its apparent catalytic efficiency kcat/Km for phytate was improved 5.3× over that of the AppA protein. The authors speculate that the C200N mutation might eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein (Lim et al., 2000) thereby modulating domain flexibility and catalytic efficiency and thermos/ability.
Three recent U.S. patents (U.S. Pat. Nos. 5,876,997, 6,110,719 and 6,190,897) by Kretz describe the sequence of the appA gene from the E. coli B strain. The AppA proteins from strains K-12 and B are identical except for two amino acid differences: K276M and T277A (residue numbering is based on the mature polypeptide). These same two amino acid changes have been discovered in the appA2 gene (Rodriguez et al., 1999).
A major source of potential genetic diversity for directed evolution studies is in nature, where phytases are found widely distributed (Wodzinski et al., “Phytase”, In Advances in Applied Microbiology, vol. 42, Academic Press, San Diego, Calif. (1996) pp. 263-302). They are found in many bacteria, fungi, and plants. Phytase activity has been detected in several bacterial sources which include the enteric bacteria E. coli (Dassa et al, 1990), Enterobacter spp. and Klebsiella spp. (Yoon et al., 1996; Greiner et al., 1997; Shah et al., 1990). Active phytase (AppA) gene variants have been successfully amplified from an E. coli strain isolated from a pig colon (Rodriguez et al., 1999) as well as from E. coli strain B (9). Besides the AppA gene product, the E. coli agp gene encodes an enzyme that has both glucose-1-phosphatase and phytase activities (Golovan et al., 2000; Kretz, U.S. Pat. No. 6,110,719). This is not entirely surprising given the fact that the two proteins share about 30% overall sequence identity including many of the amino acid residues at the conserved active site (Dassa et al., 1990). Therefore, amplification and/or cloning of the phytase variants from enteric strains (e.g., belonging to such genera as Escherichia, Salmonella, Shigella, Enterobacter and Klebsiella) is likely to generate AppA gene variants useful in the directed evolution of a commerically viable E. coli phytase (AppA) enzyme.